PROJECT SUMMARY/ABSTRACT The goal of this study is to use preclinical models to understand whether non-nicotine constituents unique to cigarette smoke (CS) contribute to differences in abuse liability between conventional cigarettes and non- combusted, alternative nicotine delivery systems (ANDs) in order to inform FDA CTP tobacco regulation. Conventional tobacco cigarettes have greater abuse liability than ANDs, such as electronic cigarettes (ECs), smokeless tobacco (ST), and nicotine replacement therapy (NRT). Non-nicotine constituents associated with CS may contribute to the greater abuse liability of cigarettes because some of these constituents (e.g., monoamine oxidase (MAO) inhibitors, volatile organic compounds (VOCs)) can mimic or enhance the effects of nicotine, or exhibit abuse liability themselves. Evaluating this hypothesis could lead to identification of new Harmful or Potentially Harmful Constituents (HPHCs) and development of addiction-related product standards that extend beyond the FDA's current focus on nicotine. Animal models are needed for this purpose, as they avoid limitations associated with human studies. Animal studies involving exposure to extracts of commercial tobacco products have been consistent with the differential abuse liability between products observed in humans. We have found that the relative abuse liability of ST extract and EC refill liquid is similar to that of nicotine alone, whereas others have found that CS extracts can produce greater addiction-related effects than nicotine alone under certain conditions. This may reflect the higher levels of addiction-relevant non-nicotine constituents in CS extracts. The proposed studies will evaluate this possibility by directly comparing the relative abuse liability of CS, ST, and EC aerosol extracts and nicotine alone (NRT analog) in state-of-the-art, FDA- recommended models for assessing abuse liability. Levels of a range of behaviorally relevant non-nicotine constituents (e.g., MAO inhibitors, acetaldehyde) will be measured and effects of isolated MAO inhibitors and VOCs will be studied to identify the specific constituents that may be responsible for observed differences in abuse liability. In Aim 1, demand for CS extract will be compared to nicotine dose-equivalent concentrations of ST extract, EC extract, and nicotine alone when each is available in isolation or under novel concurrent-choice procedures to determine relative reinforcing efficacy and substitutability of formulations. Aim 2 will compare reinforcement-enhancing and aversive effects between formulations. Aim 3 will evaluate the reinforcement- enhancing and aversive effects of isolated MAO inhibitors and VOCs when administered alone or in combination with nicotine. It is hypothesized that CS extract will have greater addiction-related effects than the other formulations due to its higher levels of behaviorally active non-nicotine constituents (e.g., MAO inhibitors, VOCs). These studies will characterize the impact of a specific tobacco product characteristic (i.e., profile of non-nicotine constituents) in tobacco abuse and inform the FDA CTP on potential compounds to consider for addition to the list of HPHCs. As such, the data will help inform specific regulatory actions of the FDA CTP.